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Background: The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear.Methods: We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation.Results: We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation.Conclusions: JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis. [ABSTRACT FROM AUTHOR]

VMXm joins the suite of operational macromolecular crystallography beamlines at Diamond Light Source. It has been designed to optimize rotation data collections from protein crystals less than 10 µm and down to below 1 µm in size. The beamline has a fully focused beam of 0.3 × 2.3 µm (vertical × horizontal) with a tuneable energy range (6-28 keV) and high flux (1.6 × 10 12  photons s -1 at 12.5 keV). The crystals are housed within a vacuum chamber to minimize background scatter from air. Crystals are plunge-cooled on cryo-electron microscopy grids, allowing much of the liquid surrounding the crystals to be removed. These factors improve the signal-to-noise during data collection and the lifetime of the microcrystals can be prolonged by exploiting photoelectron escape. A novel in vacuo sample environment has been designed which also houses a scanning electron microscope to aid with sample visualization. This combination of features at VMXm allows measurements at the physical limits of X-ray crystallography on biomacromolecules to be explored and exploited., (open access.)

Shwachman-Diamond syndrome represents a clinically and genetically heterogeneous disorder. We report on an infant with a very severe, fatal clinical course caused by biallelic EFL1 variants: c.89A>G, p.(His30Arg), and c.2599A>G, p.(Asn867Asp). Functional analysis of patient-derived B-lymphoblastoid and SV40-transformed fibroblast cell lines suggests that the compound heterozygous EFL1 variants impaired mature ribosome formation leading to compromised protein synthesis, ultimately resulting in a severe form of Shwachman-Diamond syndrome., (© 2024 The Author(s). British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

X-ray diffraction is used to study the sorption of CO and NO in two phthalocyanine nanoporous crystals (PNCs) with 4,4' bipyridine or 4,4' bipyrimidine trans coordinated to open Co 2+ sites, demonstrating how the trans coordinated ligands influence the gas sorption properties and structures of the PNCs.

Abstract: Ribosomopathy Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive inherited bone marrow failure syndrome (IBMFS) caused by mutations in the Shwachman-Bodian-Diamond syndrome gene, which is associated with an increased risk of myeloid malignancy. Tracking how hematopoietic stem cell (HSC) clonal dynamics change over time, assessing whether somatic genetic rescue mechanisms affect these dynamics, and mapping out when leukemic driver mutations are acquired is important to understand which individuals with SDS may go on to develop leukemia. In this review, we discuss how new technologies that allow researchers to map mutations at the level of single HSC clones are generating important insights into genetic rescue mechanisms and their relative risk for driving evolution to leukemia, and how these data can inform the future development of personalized medicine approaches in SDS and other IBMFSs., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Myosin motors perform many fundamental functions in eukaryotic cells by providing force generation, transport or tethering capacity. Motor activity control within the cell involves on/off switches, however, few examples are known of how myosins regulate speed or processivity and fine-tune their activity to a specific cellular task. Here, we describe a phosphorylation event for myosins of class VI (MYO6) in the motor domain, which accelerates its ATPase activity leading to a 4-fold increase in motor speed determined by actin-gliding assays, single molecule mechanics and stopped flow kinetics. We demonstrate that the serine/threonine kinase DYRK2 phosphorylates MYO6 at S267 in vitro. Single-molecule optical-tweezers studies at low load reveal that S267-phosphorylation results in faster nucleotide-exchange kinetics without change in the working stroke of the motor. The selective increase in stiffness of the acto-MYO6 complex when proceeding load-dependently into the nucleotide-free rigor state demonstrates that S267-phosphorylation turns MYO6 into a stronger motor. Finally, molecular dynamic simulations of the nucleotide-free motor reveal an alternative interaction network within insert-1 upon phosphorylation, suggesting a molecular mechanism, which regulates insert-1 positioning, turning the S267-phosphorylated MYO6 into a faster motor., (© 2024. Crown.)

α-Synuclein (αSYN), a pivotal synaptic protein implicated in synucleinopathies such as Parkinson's disease and Lewy body dementia, undergoes protein phase separation. We reveal that vesicle-associated membrane protein 2 (VAMP2) orchestrates αSYN phase separation both in vitro and in cells. Electrostatic interactions, specifically mediated by VAMP2 via its juxtamembrane domain and the αSYN C-terminal region, drive phase separation. Condensate formation is specific for R-SNARE VAMP2 and dependent on αSYN lipid membrane binding. Our results delineate a regulatory mechanism for αSYN phase separation in cells. Furthermore, we show that αSYN condensates sequester vesicles and attract complexin-1 and -2, thus supporting a role in synaptic physiology and pathophysiology., (© 2024. The Author(s).)

The growing pressure on school curricula has meant crystals and the science of crystallography have been cut from or made optional for many educational programs. This omission is a serious disservice to the history and understanding of modern sciences, given that crystallography underpins many of the greatest advancements in science over the past century, is a critical component of many modern research papers and patents, and has 29 Nobel Prizes awarded in the field. This contribution describes a simple activity to target classroom and public engagement with crystallography, using marshmallows or equivalent sweets/candy to represent atoms and cocktail sticks to represent bonds, together with examples of how crystals are studied and how they are useful. Though it has a simple basis, this activity can be extended in numerous ways to reflect the aims of the demonstrator, and a few of these are described., (© Claire Murray et al. 2024.)

Clonal tracking of cells using somatic mutations permits exploration of clonal dynamics in human disease. Here, we perform whole genome sequencing of 323 haematopoietic colonies from 10 individuals with the inherited ribosomopathy Shwachman-Diamond syndrome to reconstruct haematopoietic phylogenies. In ~30% of colonies, we identify mutually exclusive mutations in TP53, EIF6, RPL5, RPL22, PRPF8, plus chromosome 7 and 15 aberrations that increase SBDS and EFL1 gene dosage, respectively. Target gene mutations commence in utero, resulting in a profusion of clonal expansions, with only a few haematopoietic stem cell lineages (mean 8, range 1-24) contributing ~50% of haematopoietic colonies across 8 individuals (range 4-100% clonality) by young adulthood. Rapid clonal expansion during disease transformation is associated with biallelic TP53 mutations and increased mutation burden. Our study highlights how convergent somatic mutation of the p53-dependent nucleolar surveillance pathway offsets the deleterious effects of germline ribosomopathy but increases opportunity for TP53-mutated cancer evolution., (© 2023. Springer Nature Limited.)

Infectious protein crystals are an essential part of the viral lifecycle for double-stranded DNA Baculoviridae and double-stranded RNA cypoviruses. These viral protein crystals, termed occlusion bodies or polyhedra, are dense protein assemblies that form a crystalline array, encasing newly formed virions. Here, using X-ray crystallography we determine the structure of a polyhedrin from Nudiviridae. This double-stranded DNA virus family is a sister-group to the baculoviruses, whose members were thought to lack occlusion bodies. The 70-year-old sample contains a well-ordered lattice formed by a predominantly α-helical building block that assembles into a dense, highly interconnected protein crystal. The lattice is maintained by extensive hydrophobic and electrostatic interactions, disulfide bonds, and domain switching. The resulting lattice is resistant to most environmental stresses. Comparison of this structure to baculovirus or cypovirus polyhedra shows a distinct protein structure, crystal space group, and unit cell dimensions, however, all polyhedra utilise common principles of occlusion body assembly., (© 2023. The Author(s).)

The chemical modification of ribosomal RNA and proteins is critical for ribosome assembly, for protein synthesis and may drive ribosome specialisation in development and disease. However, the inability to accurately visualise these modifications has limited mechanistic understanding of the role of these modifications in ribosome function. Here we report the 2.15 Å resolution cryo-EM reconstruction of the human 40S ribosomal subunit. We directly visualise post-transcriptional modifications within the 18S rRNA and four post-translational modifications of ribosomal proteins. Additionally, we interpret the solvation shells in the core regions of the 40S ribosomal subunit and reveal how potassium and magnesium ions establish both universally conserved and eukaryote-specific coordination to promote the stabilisation and folding of key ribosomal elements. This work provides unprecedented structural details for the human 40S ribosomal subunit that will serve as an important reference for unravelling the functional role of ribosomal RNA modifications., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Neutropenia, as an isolated blood cell deficiency, is a feature of a wide spectrum of acquired or congenital, benign or premalignant disorders with a predisposition to develop myelodysplastic neoplasms/acute myeloid leukemia that may arise at any age. In recent years, advances in diagnostic methodologies, particularly in the field of genomics, have revealed novel genes and mechanisms responsible for etiology and disease evolution and opened new perspectives for tailored treatment. Despite the research and diagnostic advances in the field, real world evidence, arising from international neutropenia patient registries and scientific networks, has shown that the diagnosis and management of neutropenic patients is mostly based on the physicians' experience and local practices. Therefore, experts participating in the European Network for the Innovative Diagnosis and Treatment of Chronic Neutropenias have collaborated under the auspices of the European Hematology Association to produce recommendations for the diagnosis and management of patients across the whole spectrum of chronic neutropenias. In the present article, we describe evidence- and consensus-based guidelines for the definition and classification, diagnosis, and follow-up of patients with chronic neutropenias including special entities such as pregnancy and the neonatal period. We particularly emphasize the importance of combining the clinical findings with classical and novel laboratory testing, and advanced germline and/or somatic mutational analyses, for the characterization, risk stratification, and monitoring of the entire spectrum of neutropenia patients. We believe that the wide clinical use of these practical recommendations will be particularly beneficial for patients, families, and treating physicians., Competing Interests: FF: Advisory Board honorarium from X4 Pharmaceuticals. PEN: Consultant for X4 Pharmaceuticals. JP: Consultant to Chiesi Canada Ltd. DCD: Consultant and research support: Amgen, X4Pharma, Emendo Bio; data safety monitoring committee: Galderma, Omeros, X4Pharma, Hoffman-LaRoche, Insmed; consultant: Boerhinger-Ingelheim, Prolong,Coherus, Spectrum, Shire, Seattle Genetics. CD: Advisory Board honorarium from Gilead, Novartis, Pfizer, Rockets, Sobi. HAP: Advisory Board honorarium from X4 Pharmaceuticals. All the other authors have no conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Nirmatrelvir (PF-07321332) is a nitrile-bearing small-molecule inhibitor that, in combination with ritonavir, is used to treat infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Nirmatrelvir interrupts the viral life cycle by inhibiting the SARS-CoV-2 main protease (M pro ), which is essential for processing viral polyproteins into functional nonstructural proteins. We report studies which reveal that derivatives of nirmatrelvir and other M pro inhibitors with a nonactivated terminal alkyne group positioned similarly to the electrophilic nitrile of nirmatrelvir can efficiently inhibit isolated M pro and SARS-CoV-2 replication in cells. Mass spectrometric and crystallographic evidence shows that the alkyne derivatives inhibit M pro by apparent irreversible covalent reactions with the active site cysteine (Cys145), while the analogous nitriles react reversibly. The results highlight the potential for irreversible covalent inhibition of M pro and other nucleophilic cysteine proteases by alkynes, which, in contrast to nitriles, can be functionalized at their terminal position to optimize inhibition and selectivity, as well as pharmacodynamic and pharmacokinetic properties.

Since 2000, some thirteen quinolones and fluoroquinolones have been developed and have come to market. The quinolones, one of the most successful classes of antibacterial drugs, stabilize DNA cleavage complexes with DNA gyrase and topoisomerase IV (topo IV), the two bacterial type IIA topoisomerases. The dual targeting of gyrase and topo IV helps decrease the likelihood of resistance developing. Here, we report on a 2.8 Å X-ray crystal structure, which shows that zoliflodacin, a spiropyrimidinetrione antibiotic, binds in the same DNA cleavage site(s) as quinolones, sterically blocking DNA religation. The structure shows that zoliflodacin interacts with highly conserved residues on GyrB (and does not use the quinolone water-metal ion bridge to GyrA), suggesting it may be more difficult for bacteria to develop target mediated resistance. We show that zoliflodacin has an MIC of 4 µg/mL against Acinetobacter baumannii ( A. baumannii ), an improvement of four-fold over its progenitor QPT-1. The current phase III clinical trial of zoliflodacin for gonorrhea is due to be read out in 2023. Zoliflodacin, together with the unrelated novel bacterial topoisomerase inhibitor gepotidacin, is likely to become the first entirely novel chemical entities approved against Gram-negative bacteria in the 21st century. Zoliflodacin may also become the progenitor of a new safer class of antibacterial drugs against other problematic Gram-negative bacteria.

Background: Firefighting is among the most dangerous professions and requires exceptional physical fitness and focus while working. Patient-reported outcomes are a commonly used method to evaluate subjective health information and may be utilized by fire departments to identify the health status of firefighters and provide insight to promote their health and wellness., Objective: This study is a novel analysis of firefighters' self-reported health to potentially identify musculoskeletal dysfunction, assist in therapeutic intervention, and improve overall health and wellness., Methods: Firefighters were evaluated using seven different self-reported health surveys to assess various physical capabilities and quality of life. The questionnaires were delivered via online format and administered once to provide a snapshot of a suburban Oklahoma fire department., Results: Using the Disablement in the Physically Active Scale, 14 of the 35 firefighters answered "slight, moderate, or severe" for the pain and motion variables. Only two of the firefighters indicated no stiffness or soreness after activity on the Nirschl Phase Rating Scale. The firefighters mean rating for "energy/fatigue" via the RAND-36 was 54.14 out of 100., Conclusion: Firefighters frequently report pain, impaired motion, and soreness, indicating areas in which health and wellness interventions may be helpful. The incorporation of periodic health surveys into firefighter health and wellness programming can highlight the presence of concerns, as well as intervention effectiveness by subjective health status reporting. By combining the health surveys with aerobic and core strength exercises, fire departments may be able to monitor and improve firefighter health.

PELP1 (Proline-, Glutamic acid-, Leucine-rich protein 1) is a large scaffolding protein that functions in many cellular pathways including steroid receptor (SR) coactivation, heterochromatin maintenance, and ribosome biogenesis. PELP1 is a proto-oncogene whose expression is upregulated in many human cancers, but how the PELP1 scaffold coordinates its diverse cellular functions is poorly understood. Here we show that PELP1 serves as the central scaffold for the human Rix1 complex whose members include WDR18, TEX10, and SENP3. We reconstitute the mammalian Rix1 complex and identified a stable sub-complex comprised of the conserved PELP1 Rix1 domain and WDR18. We determine a 2.7 Å cryo-EM structure of the subcomplex revealing an interconnected tetrameric assembly and the architecture of PELP1's signaling motifs, including eleven LxxLL motifs previously implicated in SR signaling and coactivation of Estrogen Receptor alpha (ERα) mediated transcription. However, the structure shows that none of these motifs is in a conformation that would support SR binding. Together this work establishes that PELP1 scaffolds the Rix1 complex, and association with WDR18 may direct PELP1's activity away from SR coactivation., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis that initiates cytoplasmic maturation of the large ribosomal subunit. Drg1 releases the shuttling maturation factor Rlp24 from pre-60S particles shortly after nuclear export, a strict requirement for downstream maturation. The molecular mechanism of release remained elusive. Here, we report a series of cryo-EM structures that captured the extraction of Rlp24 from pre-60S particles by Saccharomyces cerevisiae Drg1. These structures reveal that Arx1 and the eukaryote-specific rRNA expansion segment ES27 form a joint docking platform that positions Drg1 for efficient extraction of Rlp24 from the pre-ribosome. The tips of the Drg1 N domains thereby guide the Rlp24 C terminus into the central pore of the Drg1 hexamer, enabling extraction by a hand-over-hand translocation mechanism. Our results uncover substrate recognition and processing by Drg1 step by step and provide a comprehensive mechanistic picture of the conserved modus operandi of AAA-ATPases., (© 2022. The Author(s).)

Background: Improving the poor prognosis of infant leukaemias remains an unmet clinical need. This disease is a prototypical fusion oncoprotein-driven paediatric cancer, with MLL (KMT2A)-fusions present in most cases. Direct targeting of these driving oncoproteins represents a unique therapeutic opportunity. This rationale led us to initiate a drug screening with the aim of discovering drugs that can block MLL-fusion oncoproteins., Methods: A screen for inhibition of MLL-fusion proteins was developed that overcomes the traditional limitations of targeting transcription factors. This luciferase reporter-based screen, together with a secondary western blot screen, was used to prioritize compounds. We characterized the lead compound, disulfiram (DSF), based on its efficient ablation of MLL-fusion proteins. The consequences of drug-induced MLL-fusion inhibition were confirmed by cell proliferation, colony formation, apoptosis assays, RT-qPCR, in vivo assays, RNA-seq and ChIP-qPCR and ChIP-seq analysis. All statistical tests were two-sided., Results: Drug-induced inhibition of MLL-fusion proteins by DSF resulted in a specific block of colony formation in MLL-rearranged cells in vitro, induced differentiation and impeded leukaemia progression in vivo. Mechanistically, DSF abrogates MLL-fusion protein binding to DNA, resulting in epigenetic changes and down-regulation of leukaemic programmes setup by the MLL-fusion protein., Conclusion: DSF can directly inhibit MLL-fusion proteins and demonstrate antitumour activity both in vitro and in vivo, providing, to our knowledge, the first evidence for a therapy that directly targets the initiating oncogenic MLL-fusion protein., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Protein synthesis is a cyclical process consisting of translation initiation, elongation, termination and ribosome recycling. The release factors SBDS and EFL1-both mutated in the leukemia predisposition disorder Shwachman-Diamond syndrome - license entry of nascent 60S ribosomal subunits into active translation by evicting the anti-association factor eIF6 from the 60S intersubunit face. We find that in mammalian cells, eIF6 holds all free cytoplasmic 60S subunits in a translationally inactive state and that SBDS and EFL1 are the minimal components required to recycle these 60S subunits back into additional rounds of translation by evicting eIF6. Increasing the dose of eIF6 in mice in vivo impairs terminal erythropoiesis by sequestering post-termination 60S subunits in the cytoplasm, disrupting subunit joining and attenuating global protein synthesis. These data reveal that ribosome maturation and recycling are dynamically coupled by a mechanism that is disrupted in an inherited leukemia predisposition disorder., (© 2022. The Author(s).)

Many cellular processes, including ribosome biogenesis, are regulated through post-transcriptional RNA modifications. Here, a genome-wide analysis of the human mitochondrial transcriptome shows that 2'-O-methylation is limited to residues of the mitoribosomal large subunit (mtLSU) 16S mt-rRNA, introduced by MRM1, MRM2 and MRM3, with the modifications installed by the latter two proteins being interdependent. MRM2 controls mitochondrial respiration by regulating mitoribosome biogenesis. In its absence, mtLSU particles (visualized by cryo-EM at the resolution of 2.6 Å) present disordered RNA domains, partial occupancy of bL36m and bound MALSU1:L0R8F8:mtACP anti-association module, allowing five mtLSU biogenesis intermediates with different intersubunit interface configurations to be placed along the assembly pathway. However, mitoribosome biogenesis does not depend on the methyltransferase activity of MRM2. Disruption of the MRM2 Drosophila melanogaster orthologue leads to mitochondria-related developmental arrest. This work identifies a key checkpoint during mtLSU assembly, essential to maintain mitochondrial homeostasis., (© 2022. The Author(s).)

Indirect somatic genetic rescue (SGR) of a germline mutation is thought to be rare in inherited Mendelian disorders. Here, we establish that acquired mutations in the EIF6 gene are a frequent mechanism of SGR in Shwachman-Diamond syndrome (SDS), a leukemia predisposition disorder caused by a germline defect in ribosome assembly. Biallelic mutations in the SBDS or EFL1 genes in SDS impair release of the anti-association factor eIF6 from the 60S ribosomal subunit, a key step in the translational activation of ribosomes. Here, we identify diverse mosaic somatic genetic events (point mutations, interstitial deletion, reciprocal chromosomal translocation) in SDS hematopoietic cells that reduce eIF6 expression or disrupt its interaction with the 60S subunit, thereby conferring a selective advantage over non-modified cells. SDS-related somatic EIF6 missense mutations that reduce eIF6 dosage or eIF6 binding to the 60S subunit suppress the defects in ribosome assembly and protein synthesis across multiple SBDS-deficient species including yeast, Dictyostelium and Drosophila. Our data suggest that SGR is a universal phenomenon that may influence the clinical evolution of diverse Mendelian disorders and support eIF6 suppressor mimics as a therapeutic strategy in SDS., (© 2021. The Author(s).)

Impaired ribosome function is the underlying etiology in a group of bone marrow failure syndromes called ribosomopathies. However, how ribosomes are regulated remains poorly understood, as are approaches to restore hematopoietic stem cell (HSC) function loss because of defective ribosome biogenesis. Here we reveal a role of the E3 ubiquitin ligase HectD1 in regulating HSC function via ribosome assembly and protein translation. Hectd1-deficient HSCs exhibit a striking defect in transplantation ability and ex vivo maintenance concomitant with reduced protein synthesis and growth rate under stress conditions. Mechanistically, HectD1 ubiquitinates and degrades ZNF622, an assembly factor for the ribosomal 60S subunit. Hectd1 loss leads to accumulation of ZNF622 and the anti-association factor eIF6 on 60S, resulting in 60S/40S joining defects. Importantly, Znf622 depletion in Hectd1-deficient HSCs restored ribosomal subunit joining, protein synthesis, and HSC reconstitution capacity. These findings highlight the importance of ubiquitin-coordinated ribosome assembly in HSC regeneration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The expression of ZAP-70 in a subset of chronic lymphocytic leukemia (CLL) patients strongly correlates with a more aggressive clinical course, although the exact underlying mechanisms remain elusive. The ability of ZAP-70 to enhance B-cell receptor (BCR) signaling, independently of its kinase function, is considered to contribute. We used RNA-sequencing and proteomic analyses of primary cells differing only in their expression of ZAP-70 to further define how ZAP-70 increases the aggressiveness of CLL. We identified that ZAP-70 is directly required for cell survival in the absence of an overt BCR signal, which can compensate for ZAP-70 deficiency as an antiapoptotic signal. In addition, the expression of ZAP-70 regulates the transcription of factors regulating the recruitment and activation of T cells, such as CCL3, CCL4, and IL4I1. Quantitative mass spectrometry of double-cross-linked ZAP-70 complexes further demonstrated constitutive and direct protein-protein interactions between ZAP-70 and BCR-signaling components. Unexpectedly, ZAP-70 also binds to ribosomal proteins, which is not dependent on, but is further increased by, BCR stimulation. Importantly, decreased expression of ZAP-70 significantly reduced MYC expression and global protein synthesis, providing evidence that ZAP-70 contributes to translational dysregulation in CLL. In conclusion, ZAP-70 constitutively promotes cell survival, microenvironment interactions, and protein synthesis in CLL cells, likely to improve cellular fitness and to further drive disease progression., (© 2021 by The American Society of Hematology.)

The mounting of microcrystals (<10 µm) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source 1 . Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise. Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

Spondyloepimetaphyseal dysplasias (SEMDs) are a heterogeneous group of disorders with variable growth failure and skeletal impairments affecting the spine and long bone epiphyses and metaphyses. Here we report on four unrelated families with SEMD in which we identified two monoallelic missense variants and one monoallelic splice site variant in RPL13, encoding the ribosomal protein eL13. In two out of four families, we observed autosomal dominant inheritance with incomplete penetrance and variable clinical expressivity; the phenotypes of the mutation-positive subjects ranged from normal height with or without hip dysplasia to severe SEMD with severe short stature and marked skeletal dysplasia. In vitro studies on patient-derived dermal fibroblasts harboring RPL13 missense mutations demonstrated normal eL13 expression, with proper subcellular localization but reduced colocalization with eL28 (p < 0.001). Cellular functional defects in fibroblasts from mutation-positive subjects indicated a significant increase in the ratio of 60S subunits to 80S ribosomes (p = 0.007) and attenuated global translation (p = 0.017). In line with the human phenotype, our rpl13 mutant zebrafish model, generated by CRISPR-Cas9 editing, showed cartilage deformities at embryonic and juvenile stages. These findings extend the genetic spectrum of RPL13 mutations causing this novel human ribosomopathy with variable skeletal features. Our study underscores for the first time incomplete penetrance and broad phenotypic variability in SEMD-RPL13 type and confirms impaired ribosomal function. Furthermore, the newly generated rpl13 mutant zebrafish model corroborates the role of eL13 in skeletogenesis. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).., (© 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).)

Background: Myofascial decompression (MFD), or cupping, and self-myofascial release (SMR) are common techniques utilized to treat soft tissue injuries and increase flexibility. MFD is a negative pressure soft tissue treatment technique using suction to manipulate the skin and underlying soft tissues. One method of SMR is a foam roller, where a patient rolls his/her bodyweight over a dense foam cylinder in a self-massaging fashion to mobilize soft tissues for the body part treated., Hypothesis/purpose: The purpose of this investigation was to examine the acute effects on hamstring flexibility and patient-rated outcome measures comparing two soft tissue treatments, 1) MFD, and 2) a moist heat pack with SMR using a foam roller in patients with diagnosed hamstring pathology., Study Design: Pilot randomized controlled trial study., Methods: Seventeen collegiate athletes [13 males (20.6+/- years; 184.9+/-cm; 90.8+/-kg) and 4 females (20.5+/-years; 167.1+/-cm; 62.7+/-kg)] with diagnosed hamstring pathology (mild strain and/or symptoms of tightness, pain, decreased strength, and decreased flexibility) were randomly assigned to receive MFD or SMR. The MFD group (n = 9) received three minutes of static treatment using six plastic-valve suction cups along the hamstrings followed by 20 repetitions of active movement with cups in place. SMR (n = 8) received 10 minutes of heat treatment over the hamstrings followed by 60 seconds of general mobilization over the entire hamstring area, and 90 seconds of targeted foam rolling on the area of most perceived tightness. Passive hamstring flexibility (ROM) and a patient-rated outcome measure [Perceived Functional Ability Questionnaire (PFAQ)] were assessed before and immediately after treatment. The Global Rating of Change measure (GROC) was administered post-intervention., Results: Passive ROM and subjective PFAQ measures for overall flexibility and flexibility of the hamstrings were significantly different from pre- to post-intervention measurements regardless of the treatment received. A significant difference was found in favor of the MFD group for the GROC values., Conclusion: The findings suggest that both treatments are beneficial in increasing hamstring length. Patients though felt an enhanced treatment effect using MFD over SMR for perceived benefits to hamstring flexibility., Levels of Evidence: Level 2., (© 2020 by the Sports Physical Therapy Section.)

Developing methods to determine high-resolution structures from micrometre- or even submicrometre-sized protein crystals has become increasingly important in recent years. This applies to both large protein complexes and membrane proteins, where protein production and the subsequent growth of large homogeneous crystals is often challenging, and to samples which yield only micro- or nanocrystals such as amyloid or viral polyhedrin proteins. The versatile macromolecular crystallography microfocus (VMXm) beamline at Diamond Light Source specializes in X-ray diffraction measurements from micro- and nanocrystals. Because of the possibility of measuring data from crystalline samples that approach the resolution limit of visible-light microscopy, the beamline design includes a scanning electron microscope (SEM) to visualize, locate and accurately centre crystals for X-ray diffraction experiments. To ensure that scanning electron microscopy is an appropriate method for sample visualization, tests were carried out to assess the effect of SEM radiation on diffraction quality. Cytoplasmic polyhedrosis virus polyhedrin protein crystals cryocooled on electron-microscopy grids were exposed to SEM radiation before X-ray diffraction data were collected. After processing the data with DIALS , no statistically significant difference in data quality was found between datasets collected from crystals exposed and not exposed to SEM radiation. This study supports the use of an SEM as a tool for the visualization of protein crystals and as an integrated visualization tool on the VMXm beamline., (© Beale et al. 2020.)

Cells possess multiple mechanisms that protect against the accumulation of toxic aggregation-prone proteins. Here, we identify a pre-emptive pathway that reduces synthesis of membrane proteins that have failed to properly assemble in the endoplasmic reticulum (ER). We show that loss of the ER membrane complex (EMC) or mutation of the Sec61 translocon causes reduced synthesis of misfolded forms of the yeast ABC transporter Yor1. Synthesis defects are rescued by various ribosomal mutations, as well as by reducing cellular ribosome abundance. Genetic and biochemical evidence point to a ribosome-associated quality-control pathway triggered by ribosome collisions when membrane domain insertion and/or folding fails. In support of this model, translation initiation also contributes to synthesis defects, likely by modulating ribosome abundance on the message. Examination of translation efficiency across the yeast membrane proteome revealed that polytopic membrane proteins have relatively low ribosome abundance, providing evidence for translational tuning to balance protein synthesis and folding. We propose that by modulating translation rates of poorly folded proteins, cells can pre-emptively protect themselves from potentially toxic aberrant transmembrane proteins., Competing Interests: Declaration of interests Authors declare no competing interests., (Copyright © 2020 MRC Laboratory of Molecular Biology. Published by Elsevier Inc. All rights reserved.)

Shwachman-Diamond syndrome (SDS) is a recessive disorder typified by bone marrow failure and predisposition to hematological malignancies. SDS is predominantly caused by deficiency of the allosteric regulator Shwachman-Bodian-Diamond syndrome that cooperates with elongation factor-like GTPase 1 (EFL1) to catalyze release of the ribosome antiassociation factor eIF6 and activate translation. Here, we report biallelic mutations in EFL1 in 3 unrelated individuals with clinical features of SDS. Cellular defects in these individuals include impaired ribosomal subunit joining and attenuated global protein translation as a consequence of defective eIF6 eviction. In mice, Efl1 deficiency recapitulates key aspects of the SDS phenotype. By identifying biallelic EFL1 mutations in SDS, we define this leukemia predisposition disorder as a ribosomopathy that is caused by corruption of a fundamental, conserved mechanism, which licenses entry of the large ribosomal subunit into translation., (© 2019 by The American Society of Hematology.)

Temperature control is a key aspect of macromolecular crystallography, with the technique of cryocooling routinely being used to mitigate X-ray-induced damage. Beam-induced heating could cause the temperature of crystals to rise above the glass transition temperature, greatly increasing the rate of damage. X-ray-induced heating of ruby crystals of 20-40 µm in size has been quantified non-invasively by monitoring the emission wavelengths of X-ray-induced fluorescence during exposure to the X-ray beam. For the beam sizes and dose rates typically used in macromolecular crystallography, the temperature rises are of the order of 20 K. The temperature changes observed are compared with models in the literature and can be used as a validation tool for future models., (open access.)

During their final maturation in the cytoplasm, pre-60S ribosomal particles are converted to translation-competent large ribosomal subunits. Here, we present the mechanism of peptidyltransferase centre (PTC) completion that explains how integration of the last ribosomal proteins is coupled to release of the nuclear export adaptor Nmd3. Single-particle cryo-EM reveals that eL40 recruitment stabilises helix 89 to form the uL16 binding site. The loading of uL16 unhooks helix 38 from Nmd3 to adopt its mature conformation. In turn, partial retraction of the L1 stalk is coupled to a conformational switch in Nmd3 that allows the uL16 P-site loop to fully accommodate into the PTC where it competes with Nmd3 for an overlapping binding site (base A2971). Our data reveal how the central functional site of the ribosome is sculpted and suggest how the formation of translation-competent 60S subunits is disrupted in leukaemia-associated ribosomopathies., Competing Interests: VK, PC, NE, KD, CH, CS, GZ, MM, SP, LW, SF, JW, AA, AF, EC, FS, HB, AW No competing interests declared, (© 2019, Kargas et al.)

The integrated stress response (ISR) is a conserved translational and transcriptional program affecting metabolism, memory, and immunity. The ISR is mediated by stress-induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, reverses the attenuation of eIF2B by phosphorylated eIF2α, protecting mice from neurodegeneration and traumatic brain injury. We describe a 4.1-angstrom-resolution cryo-electron microscopy structure of human eIF2B with an ISRIB molecule bound at the interface between the β and δ regulatory subunits. Mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB analogs in vivo and ISRIB binding in vitro. Our findings point to a site in eIF2B that can be exploited by ISRIB to regulate translation., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)